Specialized Tfh cell subsets driving type-1 and type-2 humoral responses in lymphoid tissue.

Effective antibody responses are essential to generate protective humoral immunity. Different inflammatory signals polarize T cells towards appropriate effector phenotypes during an infection or immunization. Th1 and Th2 cells have been associated with the polarization of humoral responses. However, T follicular helper cells (Tfh) have a unique ability to access the B cell follicle and support the germinal center (GC) responses by providing B cell help. We investigated the specialization of Tfh cells induced under type-1 and type-2 conditions. We first studied homogenous Tfh cell populations generated by adoptively transferred TCR-transgenic T cells in mice immunized with type-1 and type-2 adjuvants. Using a machine learning approach, we established a gene expression signature that discriminates Tfh cells polarized towards type-1 and type-2 response, defined as Tfh1 and Tfh2 cells. The distinct signatures of Tfh1 and Tfh2 cells were validated against datasets of Tfh cells induced following lymphocytic choriomeningitis virus (LCMV) or helminth infection. We generated single-cell and spatial transcriptomics datasets to dissect the heterogeneity of Tfh cells and their localization under the two immunizing conditions. Besides a distinct specialization of GC Tfh cells under the two immunizations and in different regions of the lymph nodes, we found a population of Gzmk+ Tfh cells specific for type-1 conditions. In human individuals, we could equally identify CMV-specific Tfh cells that expressed Gzmk. Our results show that Tfh cells acquire a specialized function under distinct types of immune responses and with particular properties within the B cell follicle and the GC.

IL-27 maintains cytotoxic Ly6C+ γδ T cells that arise from immature precursors.

In mice, γδ-T lymphocytes that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage during thymic development. In the periphery, these cells play a critical role in host defense and anti-tumor immunity. Unlike αß-T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ-T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that CD27+ Ly6C- cells convert into CD27+Ly6C+ cells, and these CD27+Ly6C+ cells control cancer progression in mice, while the CD27+Ly6C- cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ-T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27+Ly6C+ cells and human Vδ2+ cells, while IL-27 is dispensable for mouse CD27+Ly6C- cell and human Vδ1+ cell functions. These data reveal increased complexity within IFNγ-producing γδ-T cells, comprising immature and terminally differentiated subsets, that offer new insights into unconventional T-cell biology.

Perinatal thymic-derived CD8αß-expressing γδ T cells are innate IFN-γ producers that expand in IL-7R-STAT5B-driven neoplasms.

The contribution of γδ T cells to immune responses is associated with rapid secretion of interferon-γ (IFN-γ). Here, we show a perinatal thymic wave of innate IFN-γ-producing γδ T cells that express CD8αß heterodimers and expand in preclinical models of infection and cancer. Optimal CD8αß+ γδ T cell development is directed by low T cell receptor signaling and through provision of interleukin (IL)-4 and IL-7. This population is pathologically relevant as overactive, or constitutive, IL-7R-STAT5B signaling promotes a supraphysiological accumulation of CD8αß+ γδ T cells in the thymus and peripheral lymphoid organs in two mouse models of T cell neoplasia. Likewise, CD8αß+ γδ T cells define a distinct subset of human T cell acute lymphoblastic leukemia pediatric patients. This work characterizes the normal and malignant development of CD8αß+ γδ T cells that are enriched in early life and contribute to innate IFN-γ responses to infection and cancer.

Stressed out neutrophils drive metastasis.

Stress hormones can contribute to cancer progression, but how immune cells play a role in this process is unclear. In a recent study in Cancer Cell, He et al. showed that glucocorticoids potentiate metastasis by skewing neutrophils toward pro-tumorigenic functions.

Leukaemia exposure alters the transcriptional profile and function of BCR::ABL1 negative macrophages in the bone marrow niche.

Macrophages are fundamental cells of the innate immune system that support normal haematopoiesis and play roles in both anti-cancer immunity and tumour progression. Here we use a chimeric mouse model of chronic myeloid leukaemia (CML) and human bone marrow (BM) derived macrophages to study the impact of the dysregulated BM microenvironment on bystander macrophages. Utilising single-cell RNA sequencing (scRNA-seq) of Philadelphia chromosome (Ph) negative macrophages we reveal unique subpopulations of immature macrophages residing in the CML BM microenvironment. CML exposed macrophages separate from their normal counterparts by reduced expression of the surface marker CD36, which significantly reduces clearance of apoptotic cells. We uncover aberrant production of CML-secreted factors, including the immune modulatory protein lactotransferrin (LTF), that suppresses efferocytosis, phagocytosis, and CD36 surface expression in BM macrophages, indicating that the elevated secretion of LTF is, at least partially responsible for the supressed clearance function of Ph- macrophages.

γδ T cells control murine skin inflammation and subcutaneous adipose wasting during chronic Trypanosoma brucei infection.

African trypanosomes colonise the skin to ensure parasite transmission. However, how the skin responds to trypanosome infection remains unresolved. Here, we investigate the local immune response of the skin in a murine model of infection using spatial and single cell transcriptomics. We detect expansion of dermal IL-17A-producing Vγ6+ cells during infection, which occurs in the subcutaneous adipose tissue. In silico cell-cell communication analysis suggests that subcutaneous interstitial preadipocytes trigger T cell activation via Cd40 and Tnfsf18 signalling, amongst others. In vivo, we observe that female mice deficient for IL-17A-producing Vγ6+ cells show extensive inflammation and limit subcutaneous adipose tissue wasting, independently of parasite burden. Based on these observations, we propose that subcutaneous adipocytes and Vγ6+ cells act in concert to limit skin inflammation and adipose tissue wasting. These studies provide new insights into the role of γδ T cell and subcutaneous adipocytes as homeostatic regulators of skin immunity during chronic infection.

ß-Catenin Drives Butyrophilin-like Molecule Loss and γδ T-cell Exclusion in Colon Cancer.

Intraepithelial lymphocytes (IEL) expressing γδ T-cell receptors (γδTCR) play key roles in elimination of colon cancer. However, the precise mechanisms by which progressing cancer cells evade immunosurveillance by these innate T cells are unknown. Here, we investigated how loss of the Apc tumor suppressor in gut tissue could enable nascent cancer cells to escape immunosurveillance by cytotoxic γδIELs. In contrast with healthy intestinal or colonic tissue, we found that γδIELs were largely absent from the microenvironment of both mouse and human tumors, and that butyrophilin-like (BTNL) molecules, which can critically regulate γδIEL through direct γδTCR interactions, were also downregulated in tumors. We then demonstrated that ß-catenin activation through loss of Apc rapidly suppressed expression of the mRNA encoding the HNF4A and HNF4G transcription factors, preventing their binding to promoter regions of Btnl genes. Reexpression of BTNL1 and BTNL6 in cancer cells increased γδIEL survival and activation in coculture assays but failed to augment their cancer-killing ability in vitro or their recruitment to orthotopic tumors. However, inhibition of ß-catenin signaling via genetic deletion of Bcl9/Bcl9L in either Apc-deficient or mutant ß-catenin mouse models restored Hnf4a, Hnf4g, and Btnl gene expression and γδ T-cell infiltration into tumors. These observations highlight an immune-evasion mechanism specific to WNT-driven colon cancer cells that disrupts γδIEL immunosurveillance and furthers cancer progression.

γδ T cells turn the tables on immune-evasive colon cancer.

Why is checkpoint blockade immunotherapy still effective in tumors that are unrecognizable to CD8+ T cells? In a recent study published in Nature, de Vries et al.1 provide evidence that the lesser-known γδ T cell population may mediate beneficial responses to immune checkpoint blockade when cancer cells lose HLA expression.

PD-1 and TIM-3 differentially regulate subsets of mouse IL-17A-producing γδ T cells.

IL-17A-producing γδ T cells in mice consist primarily of Vγ6+ tissue-resident cells and Vγ4+ circulating cells. How these γδ T cell subsets are regulated during homeostasis and cancer remains poorly understood. Using single-cell RNA sequencing and flow cytommetry, we show that lung Vγ4+ and Vγ6+ cells from tumor-free and tumor-bearing mice express contrasting cell surface molecules as well as distinct co-inhibitory molecules, which function to suppress their expansion. Vγ6+ cells express constitutively high levels of PD-1, whereas Vγ4+ cells upregulate TIM-3 in response to tumor-derived IL-1ß and IL-23. Inhibition of either PD-1 or TIM-3 in mammary tumor-bearing mice increased Vγ6+ and Vγ4+ cell numbers, respectively. We found that genetic deletion of γδ T cells elicits responsiveness to anti-PD-1 and anti-TIM-3 immunotherapy in a mammary tumor model that is refractory to T cell checkpoint inhibitors, indicating that IL-17A-producing γδ T cells instigate resistance to immunotherapy. Together, these data demonstrate how lung IL-17A-producing γδ T cell subsets are differentially controlled by PD-1 and TIM-3 in steady-state and cancer.

Homologous Recombination Deficiency and Cyclin E1 Amplification Are Correlated with Immune Cell Infiltration and Survival in High-Grade Serous Ovarian Cancer.

BACKGROUND: How molecular profiles are associated with tumor microenvironment (TME) in high-grade serous ovarian cancer (HGSOC) is incompletely understood. Therefore, we analyzed the TME and molecular profiles of HGSOC and assessed their associations with overall survival (OS). METHODS: Patients with advanced-stage HGSOC treated in three Dutch hospitals between 2008-2015 were included. Patient data were collected from medical records. BRCA1/2 mutation, BRCA1 promotor methylation analyses, and copy number variations were used to define molecular profiles. Immune cells were assessed with immunohistochemical staining. RESULTS: 348 patients were categorized as BRCA mutation (BRCAm) (BRCAm or promotor methylation) (30%), non-BRCA mutated HRD (19%), Cyclin E1 (CCNE1)-amplification (13%), non-BRCAmut HRD and CCNE1-amplification (double classifier) (20%), and no specific molecular profile (NSMP) (18%). BRCAm showed highest immune cell densities and CCNE1-amplification lowest. BRCAm showed the most favorable OS (52.5 months), compared to non-BRCAmut HRD (41.0 months), CCNE1-amplification (28.0 months), double classifier (27.8 months), and NSMP (35.4 months). Higher immune cell densities showed a favorable OS compared to lower, also within the profiles. CD8+, CD20+, and CD103+ cells remained associated with OS in multivariable analysis. CONCLUSIONS: Molecular profiles and TME are associated with OS. TME differs per profile, with higher immune cell densities showing a favorable OS, even within the profiles. HGSOC does not reflect one entity but comprises different entities based on molecular profiles and TME.

NKG2D signaling regulates IL-17A-producing γδT cells in mice to promote cancer progression.

γδT cells are unconventional T cells particularly abundant in mucosal tissues that play an important role in tissue surveillance, homeostasis, and cancer. γδT cells recognize stressed cells or cancer cells through the NKG2D receptor to kill these cells and maintain normality. Contrary to the well-established anti-tumor function of these NKG2D-expressing γδT cells, we show here that, in mice, NKG2D regulates a population of pro-tumor γδT cells capable of producing IL-17A. Germline deletion of Klrk1, the gene encoding NKG2D, reduced the frequency of γδT cells in the tumor microenvironment and delayed tumor progression. We further show that blocking NKG2D reduced the capability of γδT cells to produce IL-17A in the pre-metastatic lung and that co-culture of lung T cells with NKG2D ligand-expressing tumor cells specifically increased the frequency of γδT cells. Together, these data support the hypothesis that, in a tumor microenvironment where NKG2D ligands are constitutively expressed, γδT cells accumulate in an NKG2D-dependent manner and drive tumor progression by secreting pro-inflammatory cytokines, such as IL-17A.

Assessment of CAR-T Cell-Mediated Cytotoxicity in 3D Microfluidic Cancer Co-Culture Models for Combination Therapy.

Chimeric antigen receptor (CAR)-T cell therapy is efficacious against many haematological malignancies, but challenges remain when using this cellular immunotherapy for treating solid tumours. Classical 2D in vitro models fail to recapitulate the complexity of the tumour microenvironment, whilst in vivo models, such as patient-derived xenografts, are costly and labour intensive. Microfluidic technologies can provide miniaturized solutions to assess CAR-T therapies in 3D complex preclinical models of solid tumours. Here, we present a novel microfluidic immunoassay for the evaluation of CAR-T cell cytotoxicity and targeting specificity on 3D spheroids containing cancer cells and stromal cells. Monitoring the interaction between CAR-T cells and spheroid co-cultures, we show that CAR-T cells home towards target-expressing cancer cells and elicit a cytotoxic effect. Testing CAR-T cells in combination therapies, we show that CAR-T cell cytotoxicity is enhanced with anti-PD-L1 therapy and carboplatin chemotherapy. We propose this proof-of-concept microfluidic immunoassay as a material-saving, pre-clinical screening tool for quantification of cell therapy efficacy.

The duplexity of unconventional T cells in cancer.

Unconventional T cells and their involvement in cancer are understudied in comparison to conventional T cells, but recent findings indicate that these cells play important roles in both cancer progression and inhibition. Here, we briefly review the dichotomous role of three unconventional T cell lineages: γδ T cells, MAIT cells and NKT cells. Studies using mouse models of cancer show how this unconventional trilogy interacts with cancer epithelial cells and other immune cell populations during tumour evolution. These reports highlight various potential avenues for therapeutic intervention that may be exploited for cancer immunotherapy.

Monocytes mediate Salmonella Typhimurium-induced tumor growth inhibition in a mouse melanoma model.

The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the TH 1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C+ MHCII+ monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer.

Neutrophil dynamics in the tumor microenvironment.

The tumor microenvironment profoundly influences the behavior of recruited leukocytes and tissue-resident immune cells. These immune cells, which inherently have environmentally driven plasticity necessary for their roles in tissue homeostasis, dynamically interact with tumor cells and the tumor stroma and play critical roles in determining the course of disease. Among these immune cells, neutrophils were once considered much more static within the tumor microenvironment; however, some of these earlier assumptions were the product of the notorious difficulty in manipulating neutrophils in vitro. Technological advances that allow us to study neutrophils in context are now revealing the true roles of neutrophils in the tumor microenvironment. Here we discuss recent data generated by some of these tools and how these data might be synthesized into more elegant ways of targeting these powerful and abundant effector immune cells in the clinic.

Emerging immunotherapies for metastasis.

Major advances in cancer immunotherapy have dramatically expanded the potential to manipulate immune cells in cancer patients with metastatic disease to counteract cancer spread and extend patient lifespan. One of the most successful types of immunotherapy is the immune checkpoint inhibitors, such as anti-CTLA-4 and anti-PD-1, that keep anti-tumour T cells active. However, not every patient with metastatic disease benefits from this class of drugs and patients often develop resistance to these therapies over time. Tremendous research effort is now underway to uncover new immunotherapeutic targets that can be used in patients who are refractory to anti-CTLA-4 or anti-PD-1 treatment. Here, we discuss results from experimental model systems demonstrating that modulating the immune response can negatively affect metastasis formation. We focus on molecules that boost anti-tumour immune cells and opportunities to block immunosuppression, as well as cell-based therapies with enhanced tumour recognition properties for solid tumours. We also present a list of challenges in treating metastatic disease with immunotherapy that must be considered in order to move laboratory observations into clinical practice and maximise patient benefit.

Gut γδ T cells as guardians, disruptors, and instigators of cancer.

Colorectal cancer is the third most common cancer worldwide with nearly 2 million cases per year. Immune cells and inflammation are a critical component of colorectal cancer progression, and they are used as reliable prognostic indicators of patient outcome. With the growing appreciation for immunology in colorectal cancer, interest is growing on the role γδ T cells have to play, as they represent one of the most prominent immune cell populations in gut tissue. This group of cells consists of both resident populations-γδ intraepithelial lymphocytes (γδ IELs)-and transient populations that each has unique functions. The homeostatic role of these γδ T cell subsets is to maintain barrier integrity and prevent microorganisms from breaching the mucosal layer, which is accomplished through crosstalk with enterocytes and other immune cells. Recent years have seen a surge in discoveries regarding the regulation of γδ IELs in the intestine and the colon with particular new insights into the butyrophilin family. In this review, we discuss the development, specialities, and functions of γδ T cell subsets during cancer progression. We discuss how these cells may be used to predict patient outcome, as well as how to exploit their behavior for cancer immunotherapy.